TBX1 encodes a T-box transcription factor required for cardiac development. This gene maps to the chromosome 22q11.2 region that is deleted in patients with DiGeorge syndrome/velo-cardio- facial syndrome or 22q11.2 deletion syndrome (22q11.2DS). Approximately 60% of 22q11.2DS patients have congenital heart disease that mostly affects the cardiac outflow tract. A subset of individuals with a mutation of the TBX1 gene but not a deletion has been identified and they partially phenocopy patients with 22q11.2DS. Inactivation of one allele of Tbx1 in mice results in mild defects, but inactivation of both alleles results in neonatal lethality with a persistent truncus arteriosus, in which the aorta and pulmonary trunk fail to separate. This defect also occurs in 5- 10% of 22q11.2DS patients with congenital heart disease. To understand the function of Tbx1 in mammals, we performed single cell RNA-sequencing (scRNA-seq) of cardiopharyngeal mesoderm progenitor cells within the pharyngeal apparatus. We discovered a multilineage progenitor (MLP) population that expresses genes important for forming the cardiac outflow tract as well as branchiomeric muscles of the face and neck. The MLP population expands at the expense of more differentiated populations when Tbx1 is inactivated in the cardiopharyngeal mesoderm. Our main hypothesis is that Tbx1 is required in the MLP population for progression towards more differentiated states needed for cardiac development. We propose three specific aims to test this hypothesis. In the first aim, we will perform additional scRNA-seq experiments and analyze the complete dataset to understand how the progression of MLP cells are altered in Tbx1 conditional and global mutant mouse embryos. In Aim 2, we will determine where the MLP cells are localized in the embryo. Preliminary data suggests that these cells are localized to the nascent mesenchyme of the elongating pharyngeal apparatus. We will also inactivate Tbx1 specifically within the MLP cells to determine its particular functions. In Aim 3, we will turn to functional genomic studies and will identify open and accessible chromatin for which harbors TBX1 protein binding sites using ATAC-seq and ChIP-seq from embryo tissue. Preliminary data suggests that we are able to identify direct transcriptional target genes. By these three aims, we will understand the molecular functions of TBX1 in the progression of progenitor cells to more differentiated states to build the cardiac outflow tract.